Analysis of expressed sequence tags (ESTs) using the coding sequence of human glycosyltransferases has revealed a large number of ESTs with identical as well as similar sequences. Comparison of the predicted amino acid sequence of the novel ESTs with known glycosyltransferases revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of glycosyltransferases. The likelihood that identified ESTs represent novel glycosyltransferase genes was tested by cloning and sequencing the full coding region of distinct genes followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represent genuine glycosyltransferases. In a number of cases, genomic cloning of the genes revealed that their genomic organization is identical to those of known glycosyltransferases. The results demonstrate the existence of families of homologous glycosyltransferases with rel ated functions. The existence of multiple glycosyltransferases with the same or overlapping functions may be relevant for interpreting the biological functions previously assigned to these enzymes. Kinetics and specificities of glycosyltransferases with respect to donor, acceptor, and co-factor, as well as position and anomericity of linkage in the final product were analyzed by reaction of soluble forms of the enzymes with suitable nucleotide sugar donors and glycoside acceptors. Benzyl, p-nitrophenyl, and methylumbelliferyl glycosides, as well as glycosphingolipids and glycopeptides have been used as acceptor substrates in these studies. Suitable glycosphingolipid acceptors were generated at the CCRC by chemical and enzymatic deglycosylation of purified isolates of known structure. Glycosphingolipid, glycopeptide, and glycoside acceptors and their glycosylated products were analyzed by 1- and 2-D NMR spectroscopy, comparing their spectra with those of known standards, as well as utilizing 1H-1H homonuclear (COSY, TOCSY, NOESY) and 1H-13C heteronuclear (HSQC, HMBC) methods for de novo analysis of glycosyl sequence, anomeric configuration, and linkage structure. Structure elucidation of these products with respect to newly formed linkages confirms the activity of the transferase and, by extension, the functional specificity of the gene used to express it. Two manuscripts describing this work have been published within the past year, a third has been accepted for publication, and two more are in preparation.